Immunology Field Visit Report

• WAN MAISARAH BINTI WAN ZAMRI

INTRODUCTION

On 19^th May 2015, Introduction to Immunology was organized a field trip to Veterinary Research Institute (VRI) in Ipoh, Perak. It involved all students that took Introduction to Immunology course. The trip was escorted by the course’s lecturer, Madam Nor Dini Rusli.

HISTORY OF VRI

Veterinary Research Institute (VRI), Ipoh, Perak is one section in the Division of Research and Innovation, Department of Veterinary Services, Ministry of Agriculture and Agro-based Industrian Malaysia. It was established in 1984 in temporary premises Hospital Bahagia, Tanjong Rambutan, Perak and it was formerly known as the Federal Research Laboratory. VRI today has been moved to its present location in Jalan Sultan Azlan Shah, Ipoh in 1953. It covers an area of ​​14.4 hectares. At that time, led by Director of Research VRI and assisted by 131 personnel comprising 13 Veterinary Surgeons 11 Research Officer, 2nd Assistant Veterinary Officer, 47 Laboratory Assistant, Veterinary Assistant 7 and 51 support staff. Since then, VRI has been expanded to meet the growing demand in the livestock industry.

In 2008, VRI made history as recommended by international bodies recognized as FAO’s regional reference laboratories (Regional Reference Center) for HPAI disease (bird flu) and ND (Newcastle Disease). Recognition was also given by ASEAN secretariat own and OIE. This is the highest international recognition received by VRI. This recognition is based on the ability of VRI, complete research infrastructures (BSL3, BSCL3, Unit SPF and others) as well as excellent service record in addressing the epidemic situation in the national and international levels. Therefore, the scope of activities and services that are played VRI will increase in line with current developments.

VISION AND MISSION OF VRI

Vision

Cooperation between the Veterinary Research Institute and the livestock industry by providing support services in the field of research in terms of health care practices based on proper bicultural, humane approach in the treatment of livestock and environmentally friendly use of resources.

Mission

We are committed to supporting the growth of animal industry by providing excellent services in research, diagnosis, monitoring and prevention of animal diseases through disease detection, advisory services and quality monitoring conducted by experts using the latest techniques.

FUNCTION OF VRI

• Animal health research with main focus on economically important livestock diseases and zoonotic diseases;
• Produce and commercialize vaccines, antigens, antisera, conjugates and biological materials for economically important livestock diseases and zoonotic diseases;
• Providing efficient diagnostic service for instant detection, response and effectively control animal diseases;
• Being a national reference center for the laboratories of the country;
• Providing advice, consultation, surveillance and monitoring of elimination, control and treatment of animal diseases that are new and re-emerging diseases; and
• Provide personal training to the Department of Veterinary Services, Animal Industry, the Technical Cooperation Programme Malaysia, University, College and other agencies, whether within or outside the country.

SECTIONS AND UNITS OF VRI

Sections

• Section Multimedia and Information System (MIMS)
• section of Pathology
• Section of Parasitology and Hematology
• Bacteriology Section Bird
• Bacteriology section Mammals
• Section Virology Poultry
• Section Virology Mammals
• section of Biochemistry
• Serology section
• section Immunoassay
• Section Monoclonal Antibodies
• Zoonotic section and Exotic
• Bacterial Vaccines section
• Section Vaccine Virus

Unit

• Administration and Finance Unit
• Unit Specific Pathogen Free (SPF)
• Laboratory Animal Unit
• Animal Unit Donors
• Animal Experiment Unit
• Training Unit / School

OBJECTIVES

1. To learn the immunological techniques practically.
2. To understand the purpose of immunological techniques.
3. To obtain extra knowledge in Immunology field.

METHODS OF TESTS

a) SEROLOGY SECTION

COMPLEMENT FIXATION TEST (CFT) OF BRUCELLOSIS

Complement fixation test is commonly used for serological diagnosis of Brucellosis in cattle, goats, and sheep. This test consists of two stages of reactions and requires a total of five components.

Five components of Brucellosis Complement Fixation Test

1. Serum sample : Antibody
2. Antigen : Standardized antigen diluted to working strength
3. Complement : Pooled guinea-pig serum. 1¼ Minimum Haemolytic Dose

(MHD) is used

4. Sheep red blood cell : Sheep blood collected in Alsever’s solution and stored in 4 ÌŠ C.

Immediately before use, the cell are washed and suspended as a 3% concentration in VBS.

5. Haemolysin : Rabbit anti-sheep red blood cells serum. 5 MHD is used.

For CFT indicator system, the 3% sheep red blood cells are sensitized with equal volume of 5 MHD of hemolysin. This mixture is also known as Haemolytic system.

Two stages of reactions in Brucellosis CFT

Stage 1

Serum

Stage 2

Serum

When antibody is present in the serum sample, complement is fixed which is indicated by the Haemolytic System (Sheep RBC + Haemolysin) as ‘no lysis’. Meanwhile, when antibody is absent in the serum sample, complement remains free which is indicated by Haemolytic System as ‘lysis’.

Positive	Negative
No lysis	Lysis

Materials

1. Test serum- heat inactivated in waterbath
2. Known positive serum
3. Known negative serum
4. Standardised Brucella abortus antigen for B. abortus CFT (diluted to working strength)
5. Standardised Brucella melitensis antigen for B. melitensis CFT (diluted to working strength).
6. Complement – guinea-pig serum. 1¼ (MHD) is used
7. Haemolytic system
8. Diluent- Veronal buffer saline

Equipments

1. 96-wells microtitre plates with round (U) bottom
2. Diluter machine
3. Multichannel pipette- adjustable to 150 µl
4. 25 µl and 50 µl multichannel dispenser
5. Incubator 37 ÌŠ C
6. Waterbath 37 ÌŠ C
7. Waterbath 58 ÌŠ C
8. Waterbath 62 ÌŠ C
9. Plate shaker

Test Procedure

1. Place 150 µl of undiluted serum in row H.
2. Add 25 µl of diluents to all wells in rows A to G.
3. Make 2-fold serial dilutions by transferring 25 µl undiluted serum fro row H to G onwards until row B. Discard 25 µl of the mixture from row B.
4. Add 25 µl of undiluted serum from row H to row A (serum control well). Mix and discard 25 µl.
5. Add 25 µl of standardized antigen to all wells in rows B to G.
6. Add 25 µl of diluents to row A to replace the volume of antigen.
7. Add 25 µl of 1¼ MHD of complement to all wells in rows A to G.
8. Shake the plates and incubate at 37 ÌŠ C for 30 minutes.
9. Remove paltes from incubator and add 50 µl of haemolytic system.
10. Shake the plates and incubate at 37 ÌŠ C for 15 minutes.
11. Remove plates from incubator and shake the plates, incubate for another 15 minutes.
12. Remove plate from incubator, shake and leave at room temperature for several hours tp allow unlysed cells to settle down.
13. Read the results.

Interpretation of result

Result

No haemolysis	complete fixation	4+
25% haemolysis	75% fixation	3+
50% haemolysis	50% fixation	2+
75% haemolysis	25% fixation	1+
Complete haemolysis	no fixation	0

Interpretation

≤ ½	Negative
2/2 – ¼	Doubtful
≥ 2/4	Positive

Quality control

Each batch has five controls

1. Serum anti-complementary control (A/C) in row A
2. Negative control using a known negative serum
3. Positive control using a known positive control
4. Complement control
5. Haemolytic system control

Complement control (back titration) procedure

1. Place 25 µl of VBS to wells in row H.
2. Add 25 µl of complement to all wells in rows A to G.
3. In row F, mix well and transfer 25 µl to well in row E.
4. In row E, mix well and discard 25 µl to well in row E.
5. Add 25 µl of VBS to all wells in rows D, E to F.
6. Add 25 µl of antigen to all wells.
7. Shake the plates and incubate at 37 ÌŠ C for 30 minutes.
8. Remove plates from incubator and add 50 µl of haemolytic system.
9. Shake the plates and incubate at 37 ÌŠ C for 15 minutes.
10. Remove plates from incubator and shake the plates, incubate for another 15 minutes.
11. Remove plate from incubator, shake and leave at room temperature for several hours to allow unlysed cells to settle down.

b) IMMUNOASSAY SECTION

The Immunoassay Section of VRI was created for several purposes such as :

• To diagnose veterinary diseases such as Nipah, Classical Swine Fever, Q fever, Brucellosis and etc. by using Enzyme-Linked Immuno Sorbant Assay (ELISA) technique.
• To provide theoretical and practical training courses to Department of Veterinary Services (DVS) staffs, students, and other relevant personnel.
• To provide advices to clients regarding screening and diagnostic tests on veterinary diseases via immunological and ELISA techniques.

The activities conducted in Immunoassay Section include :

• To detect serum antibody of particular disease by ELISA technique.
• Research and development on immunological and ELISA techniques.
• To train DVS staff, students and relevant personnel on the specialized skills.

The Immunoasaay Section routine work :

ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA)

The types of ELISA can be categorized into three :

• Direct ELISA
• Indirect ELISA
• Competitive ELISA

Typically, immunoassay unit at VRI performing indirect ELISA since it is the easiest technique to be conducted, conventional, but effective.

The types of tests in Immunoassay Section involves :

• Nipah on various animal species
• Brucellosis on cow
• Classical Wine Fever on pig
• Q fever on livestock
• Haemorrhagic septicaemia on cow
• Bovine Viral Disease
• Malignant Catarrhal Fever
• Infectious Bovine Rhino
• Caprine Athritis Encephalitis
• Contagious Bovine Pleural Pneumonia

The principle of ELISA include :

• ELISA is one form of Enzyme Immuno Assay (EIA).
• It is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.
• ELISA is a 96-well plate assay utilizing a reporting enzyme that is conjugated to either an antibody or an antigen.
• Enzyme activity is a direct function for the amount of the antibody bound.
• The enzyme is detected by adding substrate and looking for a change in colour that can be measured in a specialized spectrophotometer, the plate reader.

The procedure of indirect ELISA :

Buffer Preparation

1. Bicarbonate/carbonate coating buffer (100 mM)

Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:

• 3.03 g Na2CO3
• 6.0 g NaHCO3
• 1000 ml distilled water,
• pH 9.6

2. PBS:

• 1.16 g Na2HPO4
• 0.1 g KCl
• 0.1 g K3PO4
• 4.0 g NaCl (500 ml distilled water)
• pH 7.4

3. Blocking solution:

• Commonly used blocking agents are 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.

4. Wash solution:

• Usually PBS or Tris -buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

5. Antibody dilution buffer:

• Primary and secondary antibody should be diluted in 1x blocking solution to reduce non-specific binding.

Protocol

1. Dilute antigen to a final concentration of 1-20 μg/ml using PBS or Bicarbonate/carbonate coating buffer. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50μl of the antigen dilution in the top wells of the plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4°C or 2 h at room temperature.

2. Wash plate 3 times with PBS.

3. Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer, 5% non fat dry milk/PBS, per well. Alternative blocking reagents include BlockACE or BSA.

4. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more convenient, overnight at 4°C.

5. Wash the plate 3 times with PBS.

6. Add 100 μl of diluted primary antibody to each well.

7. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.

8. Wash the plate 4 times with PBS.

9. Add 100 μl of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use.

10. Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.

11. Wash the plate 5 times with PBS.

12. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipette or a multipipette.

13. After sufficient color development (if it is necessary) add 50-100μl of stop solution to the wells.

14. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.

c) BACTERIOLOGY SECTION

MAMMALS BACTERIOLOGY

Identification

Mammals bacteriology section perform isolation and identification of bacteria and fungi for further investigation and disease (diagnosis), conduct research related to the field of bacteriology animal diseases, monitoring of disease and as a reference laboratory for the veterinary laboratory or private laboratory in diagnostic bacteriology. This section is also the reference laboratory for Salmonella and Eschnerichia penserotipan coli and also testing for microbiological quality of Veterinary Public Health as milk quality testing.

Function

• Conducting research in the field of bacteriology
• Perform laboratory diagnostics of animal diseases and antimicrobial susceptibility testing ‘veterinary important bacteria
• Testing the microbiological quality of Veterinary Public Health
• Testing for microbiological quality control of milk prices for farmers
• To carry out the project impact of livestock disease problem solving where necessary
• Store and manage cultural stock of bacteria that INTERESTED veterinary
• Provide training to university students, MTCP and employees of government departments and private

BIRDS BACTERIOLOGY

Identification

Poultry bacteriology section is one section in the Veterinary Research Institute, Ipoh, which provides diagnostic services and research.In addition, the sections also engaged in the production of antigen as well as provide training to government employees and students of institutions of higher learning.

Function

• Isolation and identification of bacteria running a special for the investigation of veterinary diseases, congenital Mycoplasma sp, Avibacterium paragallinarum, Haemophilus sp, Ornithobacterium rhinotracheale (ORT) and Actinobacillus pleuropneumonia (APP)
• Conducting research on the bacteria Mycoplasma sp, Avibacterium paragallinarum, Haemophilus sp, Ornithobacterium rhinotracheale (ORT) and Actinobacillus pleuropneumonia (APP)
• Producing antigen Salmonella, Mycoplasma pen and antigen, genes and MASEN for the livestock industry
• Provide training to students of universities / colleges

BENEFITS OF FIELD TRIP

1) Deep understand about immunological techniques

During lecture session of Introduction to Immunology subject in university, we actually have been exposed about immunological techniques. Yet so, the exposure in lecture session was not completely explaining the details methods of every test because it is solely based on theory instead of practically. By joining the field trip in VRI, Ipoh, we get extra knowledge about the immunological techniques and related matters with animal health and immunology. Moreover, we can see directly a few equipments and materials involve in immunological techniques with our own eyes and get the chance to handle with them. Since VRI has experts in immunological techniques, they knew a lot about this field. They were able to explain every single thing related with the techniques until we fully understood.

2) Increase the number of enthusiasts towards immunology course

As we know, immunology is basically related with laboratory works and research. All this while, we mostly having a field trip to the farm where we need to handle with ruminants, aquaculture and poultry animals. But this time, we got new experience involved in laboratory works which is more interesting and different work environment. Undoubtedly, immunology field is a branch of study that full of its own uniqueness. I personally have long been interested in immunology and this field trip gave me opportunity to be in real situation of laboratory work particularly in immunology. Thus, it increases my passion to study in immunology.

3) May produce more female immunologist in the future

As Animal Husbandry students, we often exposed to farming work and most of occupational option related with farm. Eventhough females are not encouraged to work in farm due to the toughness, but all this while we cannot see any other choice of job that completely suitable for female who study in Animal Husbandry. So, after observing that VRI has many female workers, it seems like females has other job option which is to work in immunology laboratory one day instead of in farm because laboratory work is not as tough as working in farm. In fact, immunology field can be considered appropriate for females because it requires thoroughness corresponding to the nature of a woman conscientious. After all, this world will has more female immunologist in the future.

CONCLUSION

To sum up, the objectives of the field trip were achieved. We were able to learn the common immunological techniques used such as Complement Fixation Test (CFT) and ELISA and manage to understand them well. The information obtained from the field trip at VRI can be used in future notably for those who wants to further study in Immunology.

RECOMMENDATION

For better field trip in the future, supposedly we will be provided comfortably enough space for all of us to hear the explanation and gather up all information in order to achieve the objectives of the field trip. Since we are in large group, we should be divided into groups and each group goes to different sections to collect information and then rotate the turn of the groups to enter the sections. This ensure that everyone can clearly catch up every single words come out from the mouth of speakers. In fact, if one of the groups misses out some information, other group can collect information during their turn and then can share the information to all.

Besides that, time provided for a field trip excluding the time spent for whole journey should be enough to make sure we able to collect all information. With a long and tired journey, it is not worth to have only two to three hours of time to learn important things related with our course. It should be started early in the morning and then continue until evening with two times short breaks between sessions. This can give extra exposure to the students and able to achieve the aim of the field trip but still have time to rest for a while to refresh our body and brain.

REFERENCES

Laman Web Rasmi Institut Penyelidikan Veterinar Perkhidmatan Jabatan Veterinar. Retrieved 27^th May 2015 from http://www.dvsvri.gov.my/v3/.

Indirect ELISA Protocol. ELISA Encyclopedia. Sino Biological Incorporation. Retrieved 28^thMay 2015 from http://www.elisa-antibody.com/.